RESUMO
The objective of this work is to examine the presence of simple sequence repeat (SSR) DNA in soybean plant genotypes by Capillary Gel Electrophoresis (CGE). The SSR DNA length polymorphism in soybean determines the variation in polymerase chain reaction (PCR) product lengths. Loci were chosen where amplification produced one PCR product per genotype (M.S. Akkaya et al., 1992). The F1 hybrids of parents carrying different alleles produced two PCR products identical to the two parents. The CGE system used a 3%T,3%C polyacrylamide gel capillary with an effective length of 40 cm. The PCR products with lengths of 150 to 200 base pairs were monitored at 260 nm. The analysis time was under 50 minutes. CGE is capable of separating these PCR products by base pair number the same as conventional sequencing gel techniques. CGE offers an automated, high speed, high resolution analytical method for determining soybean SSR allele sizes as compared with the traditional methodologies.
Assuntos
Eletroforese Capilar , Glycine max/genética , Repetições de Microssatélites , Autoanálise , Mapeamento Cromossômico , DNA de Plantas/genética , Reação em Cadeia da PolimeraseRESUMO
Capillary electrophoresis (CE) is a new, high-resolution tool for the analysis of DNA restriction fragments and DNA amplified by the polymerase chain reaction (PCR). By combining many of the principles of traditional slab gel methods in a capillary format, it is possible to perform molecular size determinations of human and plant PCR amplification products and DNA restriction fragments. DNA restriction fragments and PCR products were analyzed by dynamic sieving electrophoresis (DSE) and capillary gel electrophoresis (CGE). As part of this study, sample preparation procedures, injection modes, and the use of molecular mass markers were evaluated. Optimum separations were performed using the uPage-3 (3% T, 3% C) CGE columns with UV detection at 260 nm. Membrane dialysis and ultrafiltration/centrifugation proved to be nearly equivalent methods of sample preparation. Reproducibility studies demonstrated that blunt-ended, non-phosphorylated markers (specifically allele generated markers) provide the most accurate calibration for PCR product analysis. This study demonstrates that CE offers a high-speed, high-resolution analytical method for accurately determining molecular size and/or allelic type as compared with traditional methodologies.
Assuntos
Enzimas de Restrição do DNA , DNA/análise , Eletroforese em Gel de Poliacrilamida/métodos , Reação em Cadeia da Polimerase , Bacteriófago phi X 174/genética , Sequência de Bases , Ação Capilar , DNA Viral/análise , Desoxirribonucleases de Sítio Específico do Tipo II , Eletroforese em Gel de Poliacrilamida/estatística & dados numéricos , Humanos , Dados de Sequência Molecular , Peso Molecular , Fosforilação , Plantas/genética , Reprodutibilidade dos TestesRESUMO
Commercially available capillary electrophoresis (CE) systems offer advantages over traditional slab gel methodologies. The capillary format allows the use of higher voltages (225 V/cm), which results in faster migration, higher resolution and greater efficiency without excessive heating. The ability to automate the system increases the unattended sample analysis throughput. For this study, the CE system was configured with a muPAGE 3% T, 3% C polyacrylamide gel capillary with an effective length of 40 cm and microPAGE Tris-borate urea buffer system. The analysis of DNA restriction fragments and polymerase chain reaction products, with an internal standard of Boehringer Mannheim DNA marker XI were completed in less than 70 min. All samples were analyzed at 260 nm. The data establishes automated capillary gel electrophoresis as a high-resolution, reproducible method for analysis of samples under 1000 base pairs.